Cullin-RING ligase complexes (CRLs) constitute the most abundant class of ubiquitin ligases (E3s) and regulate a host of essential cellular processes including the cell cycle. CDK inhibitors (CKIs) are critical negative regulators of the cell cycle, with a primary role to bind and inhibit CDK/cyclin complexes in the nucleus. Our earlier work showed that CUL-2 promotes the G1-S phase transition in C. elegans germ cells by negatively regulating the level of CKI-1 [Nat Cell Biol 1999,1:486]. However, we did not know whether CKI-1 is directly targeted by a CUL-2 complex (CRL2) for degradation, and if so, what substrate recognition subunit (SRS) functions in the CRL2. In our current study we identified the leucine-rich repeat protein LRR-1 as a CUL-2 interactor and showed that it functions as the SRS for a CRL2 complex to degrade CKI-1 in germ cells to allow G1 phase progression.
lrr-1 deficient animals have fewer germ cells that are significantly enlarged, with 2C DNA content, similar to
cul-2 mutants. LRR-1 is co-localized with CKI-1 in germ cell nuclei. Similar to the downregulation of CUL-2, depletion of LRR-1 causes an accumulation of CKI-1 in germ cell nuclei; and the co-depletion of CKI-1 suppresses the G1 arrest observed in
lrr-1 deficient animals. In vitro assays demonstrate that LRR-1 directly interacts with CKI-1 and induces CKI-1 degradation. Notably, we found that CRL2LRR-1 has a conserved function in human cells in targeting the CKI-1 homolog
p21. Surprisingly, human CRL2LRR1 does not regulate the cell cycle by degrading nuclear
p21. Instead, human LRR1 is localized to the cytoplasm, and controls the levels of the cytoplasmic fraction of
p21. Unlike nuclear-localized
p21, which inhibits CDK-cyclin complexes to control the cell cycle, cytoplasmic
p21 inhibits Rho kinase to control the actin cytoskeleton. siRNA knockdown of human LRR1 results in inactivation of the Rho pathway and the subsequent activation of the actin-depolymerizing protein cofilin that induces actin cytoskeletal rearrangements and cell motility. Therefore, human CRL2LRR1 normally acts to degrade cytoplasmic
p21 to maintain cells in a stationary, non-motile state. Importantly, the regulation of cell movement by LRR-1 does not appear to be conserved in C. elegans, as an analysis of the migration of distal tip cells, sex myoblasts, and touch receptor neurons in
lrr-1 mutants did not reveal significant migration defects. This data provides an interesting case study in which an alteration in the subcellular localization of the LRR-1 protein during evolution changes the cellular function (the regulation of the cell cycle vs. control of cell movement), even though the substrate remains the same (Cip/Kip CKIs).